Ocean acidification impairs seagrass performance under thermal stress in shallow and deep water.

Despite the effects of ocean acidification (OA) on seagrasses have been widely investigated, predictions of seagrass performance under future climates need to consider multiple environmental factors. Here, we performed a mesocosm study to assess the effects of OA on shallow and deep Posidonia oceanica plants. The experiment was run in 2021 and repeated in 2022, a year characterized by a prolonged warm water event, to test how the effects of OA on plants are modulated by thermal stress. The response of P. oceanica to experimental conditions was investigated at different levels of biological organization. Under average seawater temperature, there were no effects of OA in both shallow and deep plants, indicating that P. oceanica is not limited by current inorganic carbon concentration, regardless of light availability. In contrast, under thermal stress, exposure of plants to OA increased lipid peroxidation and decreased photosynthetic performance, with deep plants displaying higher levels of heat stress, as indicated by the over-expression of stress-related genes and the activation of antioxidant systems. In addition, warming reduced plant growth, regardless of seawater CO2 and light levels, suggesting that thermal stress may play a fundamental role in the future development of seagrass meadows. Our results suggest that OA may exacerbate the negative effects of future warming on seagrasses.

The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins.

In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.

Phytochemicals and antioxidant capacity in four Italian traditional maize (Zea mays L.) varieties.

Flours of four pigmented (from orange to red and dark red) local Italian corns, studied for their soluble, soluble conjugate, and insoluble-bound phenols and flavonoids, showed a prevalence of the insoluble-bound fraction (70-80%). Correlations were found between the flours antioxidant capacity, measured with CUPRAC, FRAP, and DPPH methods, and soluble phenols and flavonoids content. A correlation was also found between ascorbic acid content and flours antioxidant power. Anthocyanins were present in small amounts in the red/dark red seeds; however, acid-alcohol assays and spectral analyses of pericarp extracts indicated the presence of red-brick phlobaphenes in these varieties. Spectrophotometrically quantified total carotenoids were significantly higher in one of the local varieties (Nano); RP-HPLC analyses indicated that the local varieties contained significantly higher amounts of zeaxanthin and ß-carotene, and lower amounts of lutein, than a commercial line. Among local varieties, Nano expressed the highest levels of zeaxanthin, ß-carotene, and ß-cryptoxanthin.

Root protein profiles of two citrus rootstocks grown under iron sufficiency/deficiency conditions.

Two citrus rootstocks, one sensitive to iron deficiency (Swingle Citrumelo (SCO)) and the other tolerant (Carrizo Citrange, (CC)), were studied to characterize variation in their root protein profile induced by iron-deficient conditions. Plants of both rootstocks were grown in two different soils, one volcanic (v) and the other calcareous (c), containing 0% and 10% active Lime, respectively. To evaluate the effects of the calcareous soil on the protein accumulation of both rootstocks, the root protein profiles (SCc vs. SCv and CCc vs. CCv) were characterized by two-dimensional gel electrophoresis, thus obtaining, for the first time, a reference map of this previously uncharacterized proteome. A total of 219 spots, significantly changed in abundance, were analyzed by high-performance Liquid chromatography nano electrospray ionization tandem mass spectrometry. The identified proteins were classified according to their putative function and known biosynthetic pathways. Principal component analysis, comparing the four sets of data, indicated that each group clustered together with low variance and that CCv and CCc data sets were well differentiated, whereas SCv and SCc were similar.

Proteolytic enzymes in storage protein mobilization and cell death of the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds.

In the present manuscript, we report on the proteolytic enzymes acting in the Araucaria bidwillii megagametophyte throughout seed germination. At seed maturity the megagametophyte contains a bulk of reserves for the growing embryo, thus representing the major storage tissue of the bunya pine seed. Soon after seed germination the megagametophyte undergoes storage protein mobilization, degenerating as a no longer needed tissue by the late germinative stages. By using in-solution and in-gel assays, and mass spectrometric analyses we detected exopeptidases and proteinases differently active in this tissue at selected germinative stages, and obtained preliminary data on the nature of an endopeptidase active at the late stages. Early germination stages were characterized by aminopeptidase and aspartic, metallo and cysteine proteinase activities; carboxypeptidases and serine proteinases became highly active by the late stages. Partial sequencing of a protein responsible for late stage serine peptidase activity sensitive to the caspase-6 inhibitor, showed a set of amino acid sequences with various degrees of identity with various plant subtilisin-like serine proteinases. The participation of the early stage proteases in the storage protein mobilization and the involvement of the late stage proteases in the megagametophyte cell death are proposed and discussed.

Macro-grazer herbivory regulates seagrass response to pulse and press nutrient loading.

Coastal ecosystems are exposed to multiple stressors. Predicting their outcomes is complicated by variations in their temporal regimes. Here, by means of a 16-month experiment, we investigated tolerance and resistance traits of Posidonia oceanica to herbivore damage under different regimes of nutrient loading. Chronic and pulse nutrient supply were combined with simulated fish herbivory, treated as a pulse stressor. At ambient nutrient levels, P. oceanica could cope with severe herbivory, likely through an increase in photosynthetic activity. Elevated nutrient levels, regardless of the temporal regime, negatively affected plant growth and increased leaf nutritional quality. This ultimately resulted in a reduction of plant biomass that was particularly severe under chronic fertilization. Our results suggest that both chronic and pulse nutrient loadings increase plant palatability to macro-grazers. Strategies for seagrass management should not be exclusively applied in areas exposed to chronic fertilization since even short-term nutrient pulses could alter seagrass meadows.

The phytochelatin synthase from Nitella mucronata (Charophyta) plays a role in the homeostatic control of iron(II)/(III).

Although some charophytes (sister group to land plants) have been shown to synthesize phytochelatins (PCs) in response to cadmium (Cd), the functional characterization of their phytochelatin synthase (PCS) is still completely lacking. To investigate the metal response and the presence of PCS in charophytes, we focused on the species Nitella mucronata. A 40 kDa immunoreactive PCS band was revealed in mono-dimensional western blot by using a polyclonal antibody against Arabidopsis thaliana PCS1. In two-dimensional western blot, the putative PCS showed various spots with acidic isoelectric points, presumably originated by post-translational modifications. Given the PCS constitutive expression in N. mucronata, we tested its possible involvement in the homeostasis of metallic micronutrients, using physiological concentrations of iron (Fe) and zinc (Zn), and verified its role in the detoxification of a non-essential metal, such as Cd. Neither in vivo nor in vitro exposure to Zn resulted in PCS activation and PC significant biosynthesis, while Fe(II)/(III) and Cd were able to activate the PCS in vitro, as well as to induce PC accumulation in vivo. While Cd toxicity was evident from electron microscopy observations, the normal morphology of cells and organelles following Fe treatments was preserved. The overall results support a function of PCS and PCs in managing Fe homeostasis in the carophyte N. mucronata.

Simplified electrophoretic assay for human salivary alpha-amylase inhibitor detection in cereal seed flours.

Alpha-amylase inhibitors are antinutritional proteins largely found in cereal seeds. An in-gel assay was developed that allowed the rapid screening of these compounds in complex seed extracts. The assay was based on the electrophoretic separation of the extract proteins on starch-containing gels, followed by the detection of alpha-amylase-inhibiting proteins after incubation of the gel in an alpha-amylase solution; inhibitors were revealed by a staining method based on iodine binding to nondigested starch. The one-dimensional method can be useful to test inhibitory activity of purified proteins or to assay fractions recovered during a purification procedure. A two-dimensional (IEF x PAGE) non-denaturing system with second-dimension separation on starch-PAGE was also developed; the technique allowed the screening of complex protein mixtures for multiple inhibitory proteins. The newly developed assay method was used to test the presence of inhibitory activity in a crude extract from wheat flour, and it was validated by comparing in-gel and in-solution assays of commercially available alpha-amylase inhibitors.

Dimeric inhibitors of human salivary alpha-amylase from emmer (Triticum dicoccon Schrank) seeds.

The proteins belonging to the cereal trypsin/alpha-amylase inhibitor family are abundant water/salt-soluble flour proteins active against alpha-amylases from several seed parasites and pests and inactive against endogenous alpha-amylases. Three alpha-amylase inhibitor families have been described in cereals that vary in size and are differently expressed among Triticeae seeds. The present work investigates the presence of human salivary alpha-amylase inhibitors in emmer (Triticum dicoccon Schrank) flour. The isolation was obtained by a series of chromatography steps, and the purification progress was monitored through the inhibition of human salivary alpha-amylase activity. The purified fraction was subjected to protein sequencing by tandem mass spectrometry (MSMS) of the tryptic digests obtained after the sample separation on 2-DE. MSMS data indicated that the emmer alpha-amylase inhibitory fraction was composed of two newly identified proteins [emmer dimeric inhibitor 1 (EDI-1) and emmer dimeric inhibitor 2 (EDI-2)] sharing very high identity levels with related proteins from Triticum aestivum.

Nutrient Loading Fosters Seagrass Productivity Under Ocean Acidification.

The effects of climate change are likely to be dependent on local settings. Nonetheless, the compounded effects of global and regional stressors remain poorly understood. Here, we used CO2 vents to assess how the effects of ocean acidification on the seagrass, Posidonia oceanica, and the associated epiphytic community can be modified by enhanced nutrient loading. P. oceanica at ambient and low pH sites was exposed to three nutrient levels for 16 months. The response of P. oceanica to experimental conditions was assessed by combining analyses of gene expression, plant growth, photosynthetic pigments and epiphyte loading. At low pH, nutrient addition fostered plant growth and the synthesis of photosynthetic pigments. Overexpression of nitrogen transporter genes following nutrient additions at low pH suggests enhanced nutrient uptake by the plant. In addition, enhanced nutrient levels reduced the expression of selected antioxidant genes in plants exposed to low pH and increased epiphyte cover at both ambient and low pH. Our results show that the effects of ocean acidification on P. oceanica depend upon local nutrient concentration. More generally, our findings suggest that taking into account local environmental settings will be crucial to advance our understanding of the effects of global stressors on marine systems.

Tetraploid and hexaploid wheats express identical isoforms of nsLTP1.

Nonspecific lipid-transfer proteins (nsLTPs) have been recognized as allergens in several plant species among which are cereals important in human nutrition. In this report, we purified a 9600 +/- 1 Da protein from both soft wheat and farro bran. Mass spectrometric analyses revealed that these proteins are identical, belong to the nsLTP1 class, and have high sequence homology with nsLTP1 isolated from other cereal species. Their identification was further supported by the ability of the soft wheat nsLTP1 to transfer pyrene-labeled lipids between donor and acceptor membranes. The results are discussed in view of the increasing diffusion on the markets of bran-rich products.

NsLTP1 and NsLTP2 isoforms in soft wheat (Triticum aestivum Cv. Centauro) and farro (Triticum dicoccon Schrank) bran.

Isoforms of nonspecific lipid-transfer protein 1 (nsLTP1) and nonspecific lipid-transfer protein 2 (nsLTP2) were investigated in bran tissues isolated from caryopses of two cereal crops quite relevant for the Italian market, the cultivar Centauro of soft wheat (Triticum aestivum) and Italian emmer or farro (Triticum dicoccon Schrank). By sequential separation of the bran extracts on cation-exchange and gel filtration chromatographies, fractions containing only proteins belonging to the nsLTP1 and nsLTP2 classes were obtained. The proteins were roughly identified by SDS-PAGE and by immunoreactions in Western blotting experiments. By MALDI-MS and RP-HPLC/ESI-MS analyses we were able to show the presence of several LTP1 and LTP2 isoforms in the investigated species. Bioinformatic searches based on the determined Mr indicated that (i) two nsLTP1s already identified in T. aestivum have Mr and number of Cys residues identical to that of a 9.6 kDa protein present both in soft wheat cv. Centauro and in farro; (ii) two isoforms of nsLTP2 detected in T. aestivum have the same Mr and number of Cys residues of two 7 kDa proteins found in Centauro; and (iii) a nsLTP1 detected in Ambrosia artemisiifolia has Mr and number of Cys residues coincident to that of a 9.9 kDa protein found both in soft wheat cv. Centauro and in farro.

Trypsin/alpha-amylase inhibitors inactivate the endogenous barley/malt serine endoproteinase SEP-1.

Barley (Hordeum vulgare L.) malt contains endoproteinases belonging to all four of the commonly occurring classes, including serine proteinases. It also contains low molecular weight proteins that inhibit the activities of many of these endoproteinases, but it had never been shown that any barley or malt serine proteinases could be inhibited by any of these endogenous proteins. It is now reported that some proteins that were concentrated using an "affinity" method inhibited the activity of a malt serine endoproteinase. Two-dimensional electrophoretic and in vitro analyses showed that the inhibited enzyme was serine endoproteinase 1 (SEP-1) and that the inhibition could be quantified using a semipurified preparation of this enzyme. Amino acid sequencing and MALDI-TOF MS were used to identify the components of the partially purified inhibiting fractions. Only the "trypsin/alpha-amylase inhibitors" or chloroform/methanol (CM) proteins, most of which had truncated N and C termini, and one fragment of beta-amylase were present in the inhibitory fractions. When a CM protein fraction was prepared from barley according to traditional methods, some of its component proteins inhibited the activity of SEP-1 and some did not. This is the first report of the purification and identification of barley malt proteins that can inhibit an endogenous serine proteinase. It shows that some of the CM proteins probably play a role in controlling the activity of barley proteinases during germination, as well as possibly protecting the seed and young plant from microbes or pests.

MS-based characterization of α(s2)-casein isoforms in donkey's milk.

The primary structure of four α(s2)-casein (CN) isoforms, present as minor components in the dephosphorylated CN fraction of a milk sample collected in Eastern Sicily from an individual donkey belonging to the Ragusano breed at middle lactation stage, was determined, using the known donkey's α(s2)-CN (GenBank Acc. No. CAV00691; M(r) 26,028 Da) as reference. Proteins, with experimentally measured M(r) of 25,429, 21,939, 25,203 and 21,713 Da, were isolated by the combined use of reversed-phase high-performance liquid chromatography (RP-HPLC) and two-dimensional polyacrylamide gel electrophoresis. The major spot of each gel, corresponding to a single protein, was digested by trypsin, α-chymotrypsin and endoproteinase Glu-C. The resulting peptide mixtures were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry, and the data obtained were used for the sequence determination. The isoforms are produced from differential splicing events involving exons 4, 5 and 6 and parts of the exon 17.

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds.

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.

Proteome analysis of Citrus sinensis L. (Osbeck) flesh at ripening time.

A combination of 2-DE and LC-MSMS approaches was used to identify the differentially expressed proteome of a pigmented sweet orange (Citrus sinensis, cv. Moro) in comparison with a common cultivar (Cadenera) at ripening time. The comparison of the protein patterns of Moro and Cadenera showed 64 differential expressed protein spots. Fifty-five differentially expressed proteins were identified. Proteins were classified according to their putative function and known biosynthetic pathways. Most of the proteins related to sugar metabolism were overexpressed in Moro, while those related to stress responses were overexpressed in Cadenera. The abundance of proteins belonging to Unknown/Unnamed and Hypothetical classes could be associated to the incomplete data available on the Citrus genome. The relative abundance of Secondary metabolism and Oxidative process proteins substantiated the key role of the anthocyanin pathway in Moro, which is characterized by a strong pigmentation at ripening time. The potential role of protein differential expression between Moro and Cadenera fruits was discussed, and proteomic results were compared with the known variations of transcripts of the same fruits. The latter analyses highlighted many discrepancies, confirming the necessity to associate both proteomic and transcriptomic approaches in order to achieve a more complete characterization of the biological system.

Sequence determination of alphas1-casein isoforms from donkey by mass spectrometric methods.

Four co-eluting components, with experimentally measured M(r) of 23 658, 23 786, 24 278 and 24 406 Da, were detected by reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis in the dephosphorylated casein fraction of a milk sample collected at middle lactation stage from an individual donkey belonging to the Ragusano breed. By coupling RP-HPLC, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), enzymatic digestions, MALDI-TOF MS and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analyses, the four components were identified as donkey's alpha(s1)-CNs and their sequences completely characterized, using the known mare's alpha(s1)-CN (GenBank Acc. No. AAK83668; M(r) 23750.7 Da) as reference. The proteins with M(r) of 23 786 and 23 658 Da differ in the presence of a glutamine residue at position 83 in the full-length component and present the amino acid substitutions Q(8)-->H and H(115)-->Y with respect to the mare's alpha(s1)-CN. The other two components with M(r) 24 406 and 24 278 Da, which also differ in the presence of a glutamine residue at position 88 in the full-length component, show the insertion of the pentapeptide HTPRE between Leu(33) and the Glu(34). The two alpha(s1)-CNs bearing the pentapeptide insertion were named variants A (202 amino acids; M(r) 24 406) and A(1) (201 amino acids; M(r) 24 278), whereas the two alpha(s1)-CNs without the pentapeptide were named variants B (197 amino acids; M(r) 23 786) and B(1) (196 amino acids; M(r) 23 658).

SEP-1 - a subtilisin-like serine endopeptidase from germinated seeds of Hordeum vulgare L. cv. Morex.

Proteolysis is crucial for all living cells. It regulates protein processing, intracellular protein levels and removes abnormal or damaged proteins from the cell, working as a cellular housekeeper. By means of proteolysis, cells can control the short-lived regulatory proteins that affect processes such as signal transduction and reception, transcription, division and cellular growth. Proteolysis also furnishes amino acids for the de novo synthesis of proteins. In germinating seeds, its main role is to degrade storage proteins into small peptides and amino acids that can be used by the embryo during autotrophic growth. We have isolated and purified a serine endopeptidase, one of the many proteolytic enzymes that occur in germinated barley seeds (green malt), using chromatofocusing and DEAE-, CM-, and size-exclusion chromatographies. The enzyme, named SEP-1, has a molecular weight of 70 kDa, as estimated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. SEP-1 was detected and measured by its ability to digest gelatin in gels and to hydrolyze the synthetic substrate N-succinyl Ala-Ala-Pro-Leu p-nitroanilide. The hydrolysis of the synthetic substrate was optimal at pH 6.5 and 50 degrees C with a K(m) of 2.6 mM. The enzyme was inhibited by phenylmethylsulfonyl fluoride and p-amidinophenyl methanesulfonyl fluoride but not by any other class-specific inhibitor, suggesting it was a serine endopeptidase. Its amino acid sequence was similar to those of other plant subtilisin-like serine peptidases (EC 3.4.21), especially to the cucumisin-like group. SEP-1 was present in resting seeds, and its activity increased during germination in all of the malted barley tissues except for the endosperm, where it never occurred, suggesting that the enzyme is not likely involved in storage-protein degradation.